Characterization of Endoderm Stem Cells Derived from Murine ESCs

鼠 ESC 来源的内胚层干细胞的表征

• 批准号: 7658494
• 负责人: Jon S Odorico
• 金额: $ 18.56万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-22 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7658494
• 关键词: Address; Alveolar Cell; Animals; Back; Beta Cell; Cell Adhesion Molecules; Cell Line; Cell Separation; Cells; Characteristics; Coculture Techniques; Commit; Cultured Cells; Data; Derivation procedure; Development; ES Cell Line; Embryo; Endoderm; Endoderm Cell; Environment; Epithelial Cells; Epithelium; Generations; Genes; Germ Layers; Glucose; Goals; Growth; Growth Factor; Hepatocyte; Human; Immunodeficient Mouse; In Vitro; Injection of therapeutic agent; Insulin; Islets of Langerhans; Knowledge; Length; Liver; Lung; Magnetism; Morphology; Mus; Nodule; Organ; Organ Donor; Pancreas; Pattern; Phenotype; Population; Population Heterogeneity; Primitive Streaks; Primitive foregut structure; Production; Protocols documentation; Publishing; Replacement Therapy; Signal Transduction; Sorting - Cell Movement; Source; Stem cell transplant; Stem cells; Structure of beta Cell of islet; Teratoma; Testing; Therapeutic; Time; Tissues; To specify; Transplantation; Undifferentiated; Visceral; base; blastomere structure; cell type; embryonic stem cell; improved; in vivo; pancreas development; public health relevance; segregation; stem cell population; tumorigenic

DESCRIPTION (provided by applicant): Embryonic stem cells (ESCs) are envisioned as a source of transplantable pancreatic islets to alleviate the donor organ shortage. Despite many recent advances, protocols to differentiate ESCs to homogeneous pools of glucose-responsive insulin-secreting beta cells have yet to be developed. It is widely accepted that the production of these populations will first require generation of definitive endoderm. Thus, the successful derivation and stable propagation of endoderm-restricted stem cell lines would be a significant step forward in the development of pancreas, liver, and lung cells for cell replacement therapies and for improving our understanding of how these tissues are normally formed from this germ layer. We have developed a simple protocol, using magnetic-activated cell sorting (MACS), to isolate endoderm-committed cells from heterogeneous cultures of differentiated murine ESCs on the basis of epithelial cell adhesion molecule (EpCAM) expression. The protocol includes negative selection to remove undifferentiated ESCs and visceral endoderm cells, and positive selection to retain cells that express EpCAM. When sorted cells are placed sub-cutaneously into immunodeficient mice, small EpCAM+PDX1+ nodules highly reminiscent of embryonic pancreatic epithelium develop. Cells from these nodules can be extensively propagated in vitro and have the characteristics expected of an endodermal stem cell (EndSC) or foregut- restricted stem cell (FGSC) population, depending on the duration of growth in vivo. The EndSCs are OCT4+ and express some early endoderm-restricted genes, whereas FGSCs are OCT4- and have high expression of Sox17, Foxa2, and PDX1. EndSCs and FGSCs transplanted back into animals recapitulate the original nodule phenotype, demonstrating their stability in vitro. We hypothesize that only the OCT4+EpCAM+SSEA1- cells recovered from the MACS separation give rise to the in vivo-derived endodermal cell lines. Here, we will directly test this hypothesis by FACS-sorting OCT4+EpCAM+SSEA1- cells using an OCT4GFP ESC line. Cell lines derived from the FACS-sorted population will be compared to the existing MACS-derived EndSCs and FGSCs. We will also study the ability of EndSCs and FGSCs to differentiate in vitro to specific endoderm-derived cell types, including beta cells. To promote differentiation, we will culture cells in an embryonic pancreas environment, expose cells to relevant growth factor signals, and culture cells according to recently published protocols. These studies will contribute not only to improved differentiation efficiency of ESCs to definitive endoderm and pancreatic lineages, but also to a better understanding of germ layer segregation and early embryonic cell fate decisions. PUBLIC HEALTH RELEVANCE: Our preliminary studies have led to the development of cell lines from differentiated mouse embryonic stem cells (ESCs) that appear to be foregut endoderm-committed. These cell lines are non-tumorigenic and grow well in culture, and here we propose to elucidate the cell of origin of these endodermal cell lines and explore their ability to differentiate into pancreatic, liver, and lung cells in vitro. This knowledge will help in establishing protocols for the isolation and differentiation of human endoderm-committed cells for the production of functional cells to be used in transplantation therapies.

描述（由申请人提供）：胚胎干细胞（ESC）被设想为可移植胰岛的来源，以减轻供体器官短缺。尽管最近取得了许多进展，但尚未开发出葡萄糖反应性胰岛素β细胞的ESC与均质池库的协议。人们普遍认为，这些人群的生产将首先需要生成确定的内胚层。因此，在胰腺，肝脏和肺细胞的发展中，成功的衍生和稳定传播将是向细胞替代疗法的发展，以及提高我们对这些组织通常如何从该生殖层形成这些组织的理解，这将是向前迈出的重要一步。我们使用磁性细胞分选（MAC）开发了一个简单的方案，以根据上皮细胞粘附分子（EPCAM）表达分离内胚层的细胞从分化的鼠ESC的异质培养物中分离出来。该方案包括负面选择以去除未分化的ESC和内脏内胚层细胞，以及阳性选择以保留表达EPCAM的细胞。当分类的细胞置于免疫缺陷的小鼠中时，小的EPCAM+ PDX1+结节高度让人联想到胚胎胰腺上皮发展。这些结节的细胞可以在体外广泛传播，并具有内胚层干细胞（ENDSC）或前述限制的干细胞（FGSC）群体的特征，具体取决于体内生长持续时间。 EndScs为Oct4+，表达一些早期的内胚层限制基因，而FGSC为OCT4-，并且具有SOX17，FOXA2和PDX1的高表达。 EndScs和FGSC被移植到动物中，概括了原始的结节表型，表明它们在体外的稳定性。我们假设只有从MAC分离中回收的OCT4+EPCAM+SSEA1-细胞会产生体内衍生的内胚层细胞系。在这里，我们将使用OCT4GFP ESC线直接通过对OCT4+EPCAM+SSEA1-细胞进行FACS进行faceS分类来直接检验该假设。将源自FACS的人群得出的细胞系将与现有的MAC衍生的ENDSC和FGSC进行比较。我们还将研究EndSC和FGSC在体外区分特定内胚层细胞类型（包括β细胞）的能力。为了促进分化，我们将根据最近发表的方案在胚胎胰腺环境中培养细胞，使细胞暴露于相关的生长因子信号和培养细胞。这些研究不仅将有助于提高ESC对确定的内胚层和胰腺谱系的分化效率，而且还有助于更好地理解生殖层分离和早期的胚胎细胞命运决策。公共卫生相关性：我们的初步研究导致了来自分化小鼠胚胎干细胞（ESC）的细胞系的发展，这些细胞（ESC）似乎是腹部内胚层的。这些细胞系是非肿瘤的，在培养中生长良好，在这里我们建议阐明这些内胚层细胞系的起源细胞，并探索它们分化为体外胰腺，肝脏和肺细胞的能力。这些知识将有助于建立隔离和分化人内毒性细胞的方案，以生产用于移植疗法的功能细胞。